Synthesis method of On-DNA isoxazoline compound

Abstract

The invention discloses a synthesis method of an On-DNA isoxazoline compound, which comprises the following steps: reacting an aldehyde compound with hydroxylamine or a similar compound thereof to obtain a corresponding oxime compound, and reacting the oxime compound with an On-DNA olefin compound under the action of a metal catalyst and a reducing agent to synthesize the On-DNA isoxazoline compound. The reaction method provided by the invention provides a new method for synthesis of On-DNA isoxazoline compounds, and the reaction method is high in yield, wide in substrate universality, mild in condition, convenient to operate, low in cost and suitable for synthesis of a DNA coding compound library by using a porous plate.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA encoded compound library, and specifically relates to a method for synthesizing On-DNA isoxazoline compounds. Background Art

[0002] Professor Sydney Brenner and Richard Lerner of the Scripps Research Institute in the United States proposed the concept of DNA Encoded Library (DEL) in 1992 (reference: Proc. Natl. Acad. Sci., 1992, 89, 5381, patent: US5573905). This method connects an organic small molecule reagent with a unique sequence of DNA at the molecular level (i.e., DNA labeling of the small molecule reagent), and uses the "combine-split" strategy of combinatorial chemistry to quickly construct a huge number of compound libraries through two or more cycles. Each compound in the compound library is composed of different organic small molecule fragments and is marked by a corresponding unique base sequence of DNA. A very small amount of DNA encoded compound library is affinity screened with the target. The compound library molecules that are not adsorbed to the target will be washed away first, leaving the compound library molecules that are adsorbed to the target and then eluted. At this time, the concentration of the compound library molecules obtained is very low, and it is difficult to analyze and identify them by conventional means. However, through the unique polymerase chain reaction (PCR) of DNA, the molecular structure of the compound library can be determined by DNA. Chain Reaction, PCR for short) can replicate and amplify the DNA part of the compound library molecules that are adsorbed to the target until the amount of DNA obtained can be recognized by the DNA sequencer. The sequenced data is then decoded through the relationship table between the organic small molecule reagents created when constructing the DNA-encoded compound library and each specific DNA base sequence, and then the small molecule fragments corresponding to the specific compounds corresponding to the potential active molecules are found. These small molecule fragments are then combined together through traditional organic synthesis methods to obtain the screened target molecules, and then their physiological activity on the target is detected and confirmed.

[0003] There are three main methods for constructing DNA-encoded compound libraries. The first is the DNA-Templated Chemical Library Synthesis (DTCL) obtained by using DNA template technology mainly by Ensemble, USA. The second is the DNA-Recorded Chemical Library (DRCL) obtained by using DNA labeling technology mainly by GSK, X-Chem and Chengdu Pioneer, China. The third is the Encoded Self-Assembling Chemical Libraries (ESAC) obtained by using fragment-based drug discovery (FBDD) technology mainly by Philogen, Switzerland. Currently, the second method is the main one widely used in industry to construct DNA-encoded compound libraries. This method is simple to operate, has lower cost, and can more quickly use combinatorial chemistry methods to obtain DNA-encoded compound libraries containing a large number of compounds.

[0004] In addition to the DNA starting fragment (refer to invention patents: CN108070009A, CN109868268A), a large number of DNA tags and organic small molecule reagents that can react in a certain order are required, among which, the encoding of the DNA tag can be obtained through a certain computer program (refer to invention patent: CN107958139A), and then the primer of the specific DNA base sequence is obtained by a DNA synthesizer; the organic small molecule reagent can be obtained by using a certain computer program to screen the obtained reagent list (refer to invention patent: CN108959855A).

[0005] One of the most important tasks in the field of DEL libraries is the development of chemical reactions on DNA, referred to as On-DNA chemical reactions. Because DNA must remain stable under certain aqueous phases, pH, temperature, metal ion concentrations and inorganic salt concentrations, On-DNA chemical reactions with little DNA damage, good recovery rates and wide substrate adaptability are what is needed for large-scale application in the synthesis of DNA-encoded compound libraries. At present, there are nearly 120 types of On-DNA chemical reactions publicly reported, including aqueous phase, solid phase and DNA template reactions, and the reaction conditions of each type range from one to more than a dozen. It can be said that under the same other conditions, the more types of On-DNA chemical reactions, the richer the conditions, the better the universality, the more selectivity in the design of DNA-encoded compound libraries, the higher the success rate of synthesis of DNA-encoded compound libraries, and the richer the diversity of the obtained DNA-encoded compound libraries. In addition to these conventional chemical reactions reported publicly, due to the diverse characteristics of library synthesis, functional group transformation is often required. However, there are currently no literature or patents reporting on the synthesis method of On-DNA isoxazoline compounds through metal-catalyzed strategies.

[0006] In the construction of DNA-encoded compound libraries, isoxazoline is an important five-membered heterocyclic compound containing adjacent N and O atoms. It is commonly found in natural products. Most compounds containing this fragment have good biological activity and are widely used in new drug research and development. In the prior art, the methods for the On-DNA synthesis of isoxazoline compounds are very limited. The literature (Org. Lett., 2021, 23, 3, 908-913; Chem. Eur. J., 2023, e202203037) reported a method for obtaining On-DNA isoxazoline compounds by onDNA-α, β-unsaturated ketone and hydroxylamine. However, the raw material onDNA-α, β-unsaturated ketone of this scheme is not easy to prepare and has high cost. Moreover, since it can only form a ring with hydroxylamine, this also limits the chemical diversity of the isoxazoline DEL library.

[0007] In order to solve the limitations of the synthesis methods of On-DNA isoxazoline compounds in the construction of DNA-encoded compound libraries, it is urgent to develop a synthesis method of On-DNA isoxazoline compounds with easily available raw materials, mild reaction conditions, high yield, good substrate universality and suitable for DNA-encoded compound libraries using multi-well plates, so as to increase the diversity of DNA-encoded compound libraries. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a method for synthesizing On-DNA isoxazoline compounds, comprising the following steps:

[0009] 1) mixing 10 to 1000 equivalents of an aldehyde compound and 10 to 1000 equivalents of a hydroxylamine hydrochloride solution, reacting the mixture in a shaker, then adding 10 to 500 equivalents of chloramine-T to the reaction solution, mixing the mixture, and continuing to react the mixture in a shaker to obtain a first reaction solution;

[0010] Wherein, the aldehyde compound is selected from the following compounds:

[0011]

[0012] 2) adding 0 to 200 molar equivalents of a metal catalyst and 10 to 500 molar equivalents of a reducing agent to a mixed solution of an On-DNA olefin compound to obtain a second reaction solution, wherein the On-DNA olefin compound has a structural formula of DNA-NHCO-C=CH2, and the DNA in the On-DNA olefin compound is a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers;

[0013] 3) The first reaction solution and the second reaction solution are mixed, reacted at 20 to 50° C. for 2 to 24 hours, and then post-treated to obtain a reaction product.

[0014] The chemical formula of the reaction is as follows:

[0015]

[0016] Wherein, R1 is an amino group or a carbonyl group; R2 is an aryl group or an alkyl group.

[0017] Specifically, the concentration of the On-DNA olefin compound mixed solution is 0.1-2.0 mM.

[0018] Preferably, the concentration of the On-DNA olefin compound mixed solution is 1.0 mM.

[0019] Specifically, the On-DNA olefin compound mixed solution comprises any one or more aqueous mixed solvents selected from acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, inorganic salt buffer, organic acid buffer, and organic base buffer.

[0020] Preferably, the On-DNA olefin compound mixed solution comprises an On-DNA olefin compound and an inorganic salt buffer.

[0021] Specifically, the molar equivalent of the aldehyde compound is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents, 500 equivalents, 600 equivalents, 700 equivalents, 800 equivalents, 900 equivalents or 1000 equivalents, preferably 300 equivalents.

[0022] Specifically, the molar equivalent of the hydroxylamine hydrochloride is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents, 500 equivalents, 600 equivalents, 700 equivalents, 800 equivalents, 900 equivalents or 1000 equivalents, preferably 300 equivalents.

[0023] Specifically, the molar equivalent of the chloramine-T is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents or 500 equivalents, preferably 100 equivalents.

[0024] Specifically, the metal catalyst is one or a mixture of several of cupric chloride, palladium acetate, dihydrogen dichlorobis(di-tert-butylphosphinyl-KP) palladium acid (2-), ferrous sulfate heptahydrate, ferric chloride, magnesium chloride, stannous chloride, ytterbium trichloride hexahydrate, terpyridine ruthenium chloride, iridium trichloride, indium chloride, zirconium tetrachloride, zinc chloride, nickel chloride, cuprous chloride, cupric sulfate, cuprous iodide, cuprous bromide, cupric bromide, cuprous cyanide, cupric acetate, cupric nitrate, cuprous trifluoromethanesulfonate, and cupric tetrafluoroborate.

[0025] Preferably, the metal catalyst is cupric chloride.

[0026] Specifically, the molar equivalent of the metal catalyst is 0 equivalent, 0.1 equivalent, 0.2 equivalent, 0.3 equivalent, 0.4 equivalent, 0.5 equivalent, 0.6 equivalent, 0.7 equivalent, 0.8 equivalent, 0.9 equivalent, 1 equivalent, 2 equivalents, 3 equivalents, 4 equivalents, 5 equivalents, 6 equivalents, 7 equivalents, 8 equivalents, 9 equivalents, 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents or 200 equivalents, preferably 10 equivalents.

[0027] Specifically, the reducing agent is one of sodium ascorbate, sodium borohydride, sodium cyanoborohydride, tetrahydroxydiboron or a mixture of several thereof.

[0028] Preferably, the reducing agent is sodium ascorbate.

[0029] Specifically, the molar equivalent of the reducing agent is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents or 500 equivalents, preferably 100 equivalents.

[0030] Specifically, in step 3), the reaction temperature is 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C, preferably 20°C.

[0031] Specifically, in step 3), the reaction time is 0.5 hour, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, preferably 4 hours.

[0032] Specifically, the method is used for batch operation of multi-well plates. Preferably, the method is used for the synthesis of DNA-encoded compound libraries in multi-well plates.

[0033] The synthesis method of On-DNA isoxazoline compounds provided by the present invention realizes the cycloaddition reaction of On-DNA olefin compounds and in-situ generated oxime compounds based on the metal catalysis strategy, and is used to synthesize On-DNA isoxazoline compounds. The method provided by the present invention expands the chemical space diversity of the DNA-encoded compound library. The synthesis method disclosed by the present invention has good universality of reaction substrates, mild reaction conditions, easy availability of reactants, low cost, convenient reaction operation, high yield of products, and is suitable for the synthesis of DNA-encoded compound libraries carried out on multi-well plates. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention discloses a chemical reaction formula for preparing On-DNA isoxazoline compounds.

[0035] Figure 2 The conversion rate of the product corresponding to each aldehyde compound was obtained by LC-MS detection. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental materials involved in the present invention are all commercial reagents unless otherwise specified and can be obtained from commercial channels.

[0037] Example 1

[0038]

[0039] According to the above Figure 1 The chemical formula shown in this example shows that the synthesis steps of the On-DNA isoxazoline compound are as follows:

[0040] 1. Synthesis of On-DNA olefin compound (compound 3 below)

[0041] Refer to the reaction formula shown below:

[0042]

[0043] DNA-NH2 compound 1 (refer to the starting head fragment in patent CN108070009A) is dissolved in 250mM, pH=9.5 borate buffer, prepared into a 1.0mM concentration solution, and reacted with active lipid 2 to obtain the corresponding On-DNA olefin compound 3. After the reaction is completed, HPLC separation treatment is required, and after treatment, it can be used for the next reaction after concentration and drying.

[0044] 2. Synthesis of On-DNA isoxazoline compound (compound 5)

[0045]

[0046] First, aldehyde compound 4 (150 mM dimethylacetamide solution, 300 molar equivalents) and hydroxylamine hydrochloride (300 mM dimethylacetamide and water 1:1 mixed solution, 300 molar equivalents) were added to a 96-well plate, mixed and reacted at 20°C in a shaker for 1 hour, and then chloramine-T (250 mM dimethylacetamide and water 1:1 mixed solution, 100 molar equivalents) was added to the reaction solution, mixed and reacted at 20°C in a shaker for 1 hour, as the first reaction solution. At the same time, the product On-DNA olefin compound 3 of the previous step was dissolved in 250mM, pH = 9.5 borate buffer to prepare a 1mM concentration On-DNA mixed solution, which was dispensed into a 96-well plate, and copper chloride (25mM aqueous solution, 10 molar equivalents) and sodium ascorbate (250mM aqueous solution, 100 molar equivalents) were added respectively. After mixing, the mixture was replaced with nitrogen three times and reacted at 20°C in a shaker for 1 hour as the second reaction solution.

[0047] The first reaction solution was added to the second reaction solution, and after mixing, the mixture was replaced with nitrogen three times and reacted at 20° C. for 4 hours in a shaker. Finally, sodium diethyldithiocarbamate trihydrate (100 mM aqueous solution, 100 molar equivalents) was added to the reaction solution and reacted at 20° C. for 1 hour to remove metallic copper.

[0048] Add ethanol to precipitate:

[0049] Add 5M sodium chloride solution of 10% of the total reaction solution volume to the reaction wells of the 96-well plate, seal the film, shake and mix, add 3 times the total volume of cold anhydrous ethanol stored at -20°C, freeze in a -80°C refrigerator for 2 hours, then take out and centrifuge at 4°C with a centrifugal force of 4000G for 30 minutes, remove the supernatant, dissolve the precipitate in deionized water and freeze-dry at -40°C in a vacuum to obtain the product, detect OD by an enzyme marker to confirm the recovery rate, and detect LC-MS to confirm the conversion rate of each small molecule. The results are shown in Figure 2 , which shows the conversion rate corresponding to each aldehyde compound. It can be seen that the reaction conditions have a good conversion rate in both 5-membered and 6-membered aromatic aldehydes, and when the aldehyde is an aliphatic aldehyde, the method provided in the present application also has a good application effect.

[0050] To sum up, the above embodiments and drawings are only some representative embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for synthesizing an On-DNA isoxazoline compound, characterized in that: The following steps are involved: 1) mixing 10 to 1000 equivalents of an aldehyde compound and 10 to 1000 equivalents of a hydroxylamine hydrochloride solution, reacting the mixture in a shaker, then adding 10 to 500 equivalents of chloramine-T to the reaction solution, mixing the mixture, and continuing to react the mixture in a shaker to obtain a first reaction solution; Wherein, the aldehyde compound is selected from the following compounds: 2) adding 0 to 200 molar equivalents of a metal catalyst and 10 to 500 molar equivalents of a reducing agent to a mixed solution of an On-DNA olefin compound to obtain a second reaction solution, wherein the On-DNA olefin compound has a structural formula of DNA-NHCO-C=CH2, and the DNA in the On-DNA olefin compound is a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers; 3) The first reaction solution and the second reaction solution are mixed, reacted at 0 to 100° C. for 0.5 to 24 hours, and then post-treated to obtain a reaction product.

2. The method for synthesizing an On-DNA isoxazoline compound according to claim 1, characterized in that: The concentration of the On-DNA olefin compound mixed solution is 0.1-2.0 mM.

3. The method for synthesizing an On-DNA isoxazoline compound according to claim 1, characterized in that: The On-DNA olefin compound mixed solution comprises any one or more aqueous mixed solvents selected from acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, inorganic salt buffer, organic acid buffer, and organic base buffer.

4. The method for synthesizing an On-DNA isoxazoline compound according to claim 1, characterized in that: The molar equivalent of the aldehyde compound is 300 equivalents.

5. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: The molar equivalent of the hydroxylamine hydrochloride is 300 equivalents.

6. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: The molar equivalent of the chloramine-T is 100 equivalents.

7. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: The metal catalyst is one or a mixture of the following: cupric chloride, palladium acetate, dihydrogen dichlorobis(di-tert-butylphosphinyl-KP) palladium acid (2-), ferrous sulfate heptahydrate, ferric chloride, magnesium chloride, stannous chloride, ytterbium trichloride hexahydrate, terpyridine ruthenium chloride, iridium trichloride, indium chloride, zirconium tetrachloride, zinc chloride, nickel chloride, cuprous chloride, cupric sulfate, cuprous iodide, cuprous bromide, cupric bromide, cuprous cyanide, cupric acetate, cupric nitrate, cuprous trifluoromethanesulfonate, and cupric tetrafluoroborate.

8. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: The molar equivalent of the metal catalyst is 10 equivalents.

9. The method for synthesizing an On-DNA isoxazoline compound according to claim 1, characterized in that: The reducing agent is one or a mixture of sodium ascorbate, sodium borohydride, sodium cyanoborohydride, and tetrahydroxydiboron.

10. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: The molar equivalent of the reducing agent is 100 equivalents.

11. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: In step 3), the reaction temperature is 20°C.

12. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: In step 3), the reaction time is 4 hours.

13. The method for synthesizing On-DNA isoxazoline compounds according to claim 1, characterized in that: The method is used for batch operation of multiwell plates.

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